Horseradish peroxidase is widely used as one of enzymes for detection in various tests such as enzyme-linked immunosorbent assay (ELISA), immunohistostaining method, Southern blotting method, and western blotting method. Moreover, the peroxidase has also been widely used as an enzyme for clinical inspection kits recently.
In general, peroxidases are widely present in the plant kingdom such as white radish (Raphanus sativus), sweet potato (Ipomoea batatas), wheat (Triticum spp.), Japanese horseradish (Eutrema japonica), and horseradish. For the reason that the peroxidase content in horseradish is high, or other reasons, horseradish is preferably used in industrial productions.
Meanwhile, horseradish peroxidase includes multiple enzymes such as acidic, neutral, and basic isozymes. Furthermore, the peroxidase content and the composition ratio of these isozymes greatly vary depending on: the properties of the soil where the horseradish is cultivated, the type and the amount of a fertilizer added, weather, harvesting time, and so forth. In addition, in a method for producing a peroxidase, a plant is destroyed and the peroxidase is purified from a great variety of contaminants. From the foregoing, the quality of a peroxidase purified from a horseradish is not always the same. In other words, most of horseradish peroxidase products used widely at present are mixtures of a large number of isozymes. In the majority of cases, the ratio is different from lot to lot. When such a horseradish peroxidase product is used to perform various measurements, for example, ELISA, a serious problem occurs that it is difficult to obtain stable measurement results because the results vary among production lots.
Moreover, horseradish that serves as the raw material requires a long period for cultivation and accordingly has a yield that varies depending on the weather. In addition, recently, a concern has been growing regarding a situation that horseradish supply might become short for reasons of: low cultivation efficiency, alternate farming to cereals for bioethanol having a higher demand, and the like. Hence, there is a great potential need for horseradish peroxidases that can be supplied steadily.
A conceivable method for overcoming the aforementioned problems of horseradish peroxidase is mass production by microorganisms using genetic recombination techniques. The use of microorganisms not only enables mass culture within a short period, but also enables steady supply without influence from the weather. Moreover, the use of genetic recombination techniques enables a single expression of a target peroxidase in a large amount. This makes it possible to avoid a problem of isozyme contamination.
The DNA sequence and the amino acid sequence of peroxidase C1a, one of main isozymes of horseradish peroxidase, have been revealed (see NPL 1). Moreover, using a gene encoding peroxidase C1a, the expression in Escherichia coli, yeast, and tobacco (Nicotiana tabacum) plant cells has been studied. However, the expression amount is as small as 0.11 mg/L in Escherichia coli (see NPL 2), 5.3 mg/L in yeasts (see NPL 3), and 3 mg/L in tobacco plant cells (see NPL 4). The productivity is quite low and impractical. From the foregoing, it has been desired to develop a recombinant organism capable of mass production of horseradish peroxidase, and a method for producing horseradish peroxidase using the recombinant organism.